Pharmaceutical parenteral formulation containing carglumic acid

ABSTRACT

The present invention relates to a pharmaceutical formulation suitable for parenteral administration containing carglumic acid and a buffering agent having a pKa from 5.5 to 9.0 at 25° C.; according to an embodiment, the buffering agent may have a pKa from 7.5 to 8.5, preferably a pKa of about 8.07, such as trometamol. The formulation may also contain at least one bulking agent, such as mannitol. The invention also includes a method for manufacturing a lyophilised sterile formulation by freeze-drying a water solution containing carglumic acid, a buffering agent having a pKa from 5.5 to 9.0 at 25° C., preferably from 7.5 to 8.5, and optionally a bulking agent to obtain a freeze-dried powder.

The present invention relates to a pharmaceutical formulation suitablefor parenteral administration containing carglumic acid and a bufferingagent having a pK_(a) from 5.5 to 9.0 at 25° C.; according to anembodiment, the buffering agent may have a pK_(a) from 7.5 to 8.5,preferably a pK_(a) of about 8.07, such as trometamol. The formulationmay also contain at least one bulking agent, such as mannitol. Theinvention also includes a method for manufacturing a lyophilised sterileformulation by freeze-drying a water solution containing carglumic acid,a buffering agent having a pK_(a) from 5.5 to 9.0 at 25° C., preferablyfrom 7.5 to 8.5, and optionally a bulking agent to obtain a freeze-driedpowder.

BACKGROUND

Carglumic acid, whose chemical formula is reported below, is an activeprinciple that is used for the treatment of hyperammonaemia (high bloodlevels of ammonia).

It is marketed in the EU under the trademark Carbaglu® in the form oftablets that must be dispersed in water and ingested immediately oradministered by fast push through a syringe via a nasogastric tube,generally in case of hospitalised patients or patients who are not ableto swallow.

Carglumic acid is highly hygroscopic and suffers some instabilityproblems. For instance, unopened Carbaglu® containers should be tightlyclosed and stored at 2 to 8° C. After its first opening, the containermust be stored at a temperature above the refrigerated temperature butbelow 30° C.; furthermore, any unused tablet must be discarded after onemonth from the first opening.

Processes for manufacturing tablets containing carglumic acid by directcompression are disclosed in EP2777696 and CN105056246.

Due to long term instability of carglumic acid once solubilised,nowadays there are not ready-to-use intravenous or, anyway, injectablesterile formulations on the market containing carglumic acid that couldbe used for emergency purposes.

DESCRIPTION OF THE INVENTION

The purpose of the present invention is providing a new solidpharmaceutical formulation having a higher content of carglumic acidthan Carbaglu®, an improved dissolution profile and improved stabilityand which, once reconstituted with water, could be administeredintravenously, for instance by infusion and/or injection, particularlywhen a rapid therapeutic effect is needed.

Another purpose of the invention is providing a method for manufacturingsaid pharmaceutical formulation that does not affect the stability ofthe active principle itself.

Another purpose of the invention is providing a method for manufacturingsaid pharmaceutical formulation which may stabilise the obtained steriledosage form when stored at 2-8° C.

Another purpose of the invention is providing a method for manufacturingsaid pharmaceutical formulation which may stabilise the obtained steriledosage form when stored at 25° C.

Which technical problems have been solved by means of a pharmaceuticalformulation obtained through a freeze-drying process as discussed below.

Freeze-drying, also known as lyophilisation, is a dehydration processtypically used to formulate into a dosage form a perishable or unstableactive principle. Freeze-drying works by freezing a water mixture of theactive principle together with one or more physiologically acceptableexcipients and then reducing the surrounding pressure to allow thefrozen water to sublimate directly from the solid phase to the gasphase.

In order to allow an effective and industrially applicable freeze-dryingprocess, the water mixture to be frozen must also be chemically andphysically stable and, possibly, should be a clear water solution(crystallization or precipitation should be avoided). An intravenouslyadministrable formulation must also be a clear and sterile water-basedsolution.

The applicant has performed several tests in order to find the mostappropriate conditions for obtaining a clear solution of carglumic acidat high concentrations, which included the use of HCl 0.5 M, NaOH 0.5 Mor a phosphate and dextrose buffer; such attempts however failed, sinceno clear and stable solution of carglumic acid could be achieved.

Nevertheless, as it shall be appreciated from the attached experimentalsection, trometamine (also known as TRIS), which is a buffering agenthaving a pK_(a) from 7.5 and 9.0 at 25° C., surprisingly providedexcellent results even at high carglumic acid concentrations. Inaddition, excellent results in terms of manufacturability and productstability were also surprisingly obtained using mannitol as the bulkingagent.

The subject-matter of the present invention is thus represented by apharmaceutical formulation containing carglumic acid or apharmaceutically acceptable salt or derivative thereof and a bufferingagent having a pK_(a) from 5.5 to 9.0 at 25° C., preferably a pK_(a)from 7.5 and 8.5 at 25° C., more preferably a pK_(a) of about 8.07, suchas trometamol.

According to an embodiment of the invention, the formulation may alsocontain at least one bulking agent, such as, but not limited tomannitol. Other bulking agents that may be used in the formulation ofthe present invention are lactose, threalose, glycine, dextrane,sucrose, glucose, fructose, sorbitol, inositol.

The pharmaceutical formulation according to the present invention mayalso contain one or more physiologically acceptable excipients inaddition to said buffering agent and said bulking agent.

According to a further embodiment, the carglumic acid:trometamol molarratio is from 1:1 and 1:2.5, preferably about 1:2.

According to a further embodiment, the weight ratio between carglumicacid and the bulking agent, such as mannitol, is from 25:32 and 25:50,preferably about 25:40.

According to a further embodiment, the pharmaceutical formulation of thepresent invention may be a powder that should be reconstituted in waterbefore use.

According to another embodiment, each dosage form may contain from 400to 600 mg of carglumic acid, preferably about 500 mg of carglumic acid;in case of dosage forms intended to be used by patients belonging to thepaediatric population, each dosage form may contain from 25 to 200 mg ofcarglumic acid, preferably about 50 mg of carglumic acid.

According to a further embodiment, the pharmaceutical formulation of thepresent invention may be a water solution, namely either the mixturethat will be subjected to freeze-drying in order to provide theabove-mentioned lyophilised product or the solution to be administeredintravenously once reconstituted with water; such a water solutionpreferably has a carglumic acid concentration higher than 2%weight/volume, preferably higher than or equal to 2.5% weight/volume.

According to the best embodiment of the invention, the formulationcontains carglumic acid, trometanol and mannitol, the carglumicacid:trometamol molar ratio is from 1:1 and 1:2.5, preferably about 1:2and the carglumic acid:mannitol weight ratio is from 25:32 and 25:50,preferably about 25:40

The subject-matter of the present invention is also represented by amethod for manufacturing a powder, which comprises subjecting tofreeze-drying a water solution containing carglumic acid, a bufferingagent having a pK, from 7.5 and 9.0 at 25° C. (such as trometamol) and abulking agent (such as mannitol) to obtain a freeze-dried powder.

A further subject-matter of the invention is then represented by amethod for treating hyperammonaemia which comprises administering thepresent pharmaceutical formulation to a human in need of such atreatment.

Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference;thus, the inclusion of such definitions herein should not be construedto represent a substantial difference over what is generally understoodin the art.

In particular, the term “physiologically acceptable excipient” hereinrefers to a substance devoid of any pharmacological effect of its ownand which does not produce adverse reactions when administered to amammal, preferably a human. Physiologically acceptable excipients arewell known in the art and are disclosed, for instance in the Handbook ofPharmaceutical Excipients, sixth edition 2009, herein incorporated byreference.

The term “Pharmaceutically acceptable salts or derivatives” hereinrefers to those salts or derivatives which possess the biologicaleffectiveness and properties of the salified or derivatized compound andwhich and which do not produce adverse reactions when administered to amammal, preferably a human. The pharmaceutically acceptable salts may beinorganic or organic salts; examples of pharmaceutically acceptablesalts include but are not limited to: carbonate, hydrochloride,hydrobromide, sulphate, hydrogen sulphate, citrate, maleate, fumarate,trifluoroacetate, 2-naphthalenesulphonate, and para-toluenesulphonate.Further information on pharmaceutically acceptable salts can be found inHandbook of pharmaceutical salts, P. Stahl, C. Wermuth, WILEY-VCH,127-133, 2008, herein incorporated by reference. The pharmaceuticallyacceptable derivatives include the esters and the ethers.

The term “bulking agent” herein refers to a physiologically acceptableexcipient that increases the volume or the weight of a pharmaceuticalformulation keeping its utility or functionality intact.

The term “buffering agent” herein refers to a weak acid or base used tomaintain the acidity (pH) of a water solution near a chosen value afterthe addition of another acid or base.

The term “IV” herein means intravenous injection or intravenouslyinjectable.

The term “ICH conditions” herein refers to the thermohygrometricconditions of storage of Drug Products that are intended for alreadymarketed products or submissions of new Marketing Authorizations (MA),outlined by the International Council on Harmonisation (ICH) guidelines.

The terms “approximately” and “about” herein refers to the range of theexperimental error, which may occur in a measurement.

The terms “comprising”, “having”, “including” and “containing” are to beconstrued as open-ended terms (i.e. meaning “including, but not limitedto”) and are to be considered as providing support also for terms as“consist essentially of”, “consisting essentially of”, “consist of” or“consisting of”.

The terms “consist essentially of”, “consisting essentially of” are tobe construed as a semi-closed terms, meaning that no other ingredientswhich materially affects the basic and novel characteristics of theinvention are included (optional excipients may thus included).

The terms “consists of”, “consisting of” are to be construed as closedterms.

The term “paediatric population” herein refers to that part of thepopulation from birth to eighteen years.

Experimental Section

Preliminary Solubility Trials

Preliminary solubility trials have been performed using carglumic acidsolutions buffered at pH from 5.0 to 5.7 with NaOH (0.5 M) or phosphate;the test were not successful since the solutions were not stable andgave an unknown degradation product (with molecular ion at 159[m+H]+detected in HPLC-mass-spectrometry) not present in the solution ofcarglumic acid in water.

Carglumic acid solution in HCl 0.5 M also did not result stable up to 24hours, since two known impurities were detected in concentrations abovethe accepted limits.

After the failure of the preliminary tests with HCl, NaOH or phosphate,two different approaches were considered to develop a freeze-driedproduct of carglumic acid for injection (500 mg/vial), namely:

-   -   freeze-dried formulation containing carglumic acid and bulking        agent to be reconstituted with a diluent containing trometamol        as buffering agent;    -   freeze-dried formulation containing carglumic acid, trometamol        and bulking agent.

Initial solubility trials were set up to understand which manufacturingand formulation approach could be more suitable.

a. Carglumic Acid in Water Solution

100 ml of solution were prepared dissolving 2.5 g of carglumic acid inwater. A white suspension was obtained. The suspension was stirred for30 minutes without obtaining a clear solution. The solution was heatedat about 45° C. A complete solution was obtained at pH=1.9

Carglumic Acid in Water Solution with Trometamol

100 ml of solution were prepared dissolving 5 g of trometamol in water;2.5 g of carglumic acid were then added. A clear solution was obtainedimmediately at room temperature, with pH=8.2

Both formulations were observed after 24 hours storage at RT, 5° C. and−20° C. The results are summarized in table 1.

TABLE 1 Room Formulation temperature 5° C. −20° C.* Carglumic acid inwater Clear Crystalline Powder solution solution precipitate precipitateCarglumic acid in water Clear Clear Clear solution with trometamolsolution solution solution *= after thawing

These results demonstrated that the presence of a buffering agent havinga pK_(a) from 7.5 and 9.0 at 25° C., such as trometamol, is essential toobtain a clear and stable lyophilisable solution.

Bulking Agent Selection

The first lyophilisation trial was performed in order to select thebulking agent. Laboratory-scale batches (2000 ml) of placebo solutionscontaining mannitol and lactose as bulking agents were prepared. 20 mlvials were prepared and lyophilised for each formulation. Thequali-quantitative compositions of the formulations are reported intable 2.

TABLE 2 Component 1 ml 1 vial (20 ml) Formula for mannitol placeboMannitol 40 mg 800 mg Trometamol 32 mg 640 mg HCl 37% For adjustment topH = 8 Water for injection Qs to 1 ml Qs to 20 ml Formula for lactoseplacebo Lactose 40 mg 800 mg Trometamol 32 mg 640 mg HCl 37% Foradjustment to pH = 8 Water for injection Qs to 1 ml Qs to 20 ml

The appearance of the mannitol formulation at the end of lyophilisationcycle resulted in a white quite compact cake; the lactose formulationappearance was a dark yellow melted cake. Mannitol was thus selected asthe preferred bulking agent for carglumic acid formulation development.

Bulk Solution Formulation Screening for pH and Osmolality

Different formulations of carglumic acid (API) and trometamol (TRIS)molar ratio and bulking agent concentrations were prepared in order tomeasure pH and osmolality at the end of solutions' preparation. Thequali-quantitative compositions of the formulations are reported intable 3.

TABLE 3 API: Bulking Formu- TRIS Bulking API:TRIS agent Osmolalitylation mg agent mg molar ratio (g/100 ml) pH (Osmol/kg) A 25:32 40 1:24% 6.55 0.582 B 25:50 40 1:3 4% 8.31 0.763 C 25:16 40 1:1 4% 4.02 0.477D 25:40 40   1:2.5 4% 7.88 0.671 E 25:35 40   1:2.2 4% 7.56 0.615 F25:32 20 1:2 2% 6.58 0.465 G 25:32 10 1:2 1% 4.35 0.386 H 25:32 5 1:20.5%   4.77 0.316 I 25:32 — 1:2 — 4.88 0.289 L 25:33 10   1:2.1 1% 6.830.399 M 25:33 5   1:2.1 0.5%   6.84 0.358 N 25:33 —   1:2.1 — 7.04 0.341

Since the formulation must be intravenously injectable, the pH targetshould be in the range of 6.5-7.5 while osmolality should be in therange of 0.290-0.600 Osm/Kg when the cake is reconstituted with waterfor injection. In view of their osmolality and pH, formulations A, F, L,and N were selected for a lyophilisation trial. The results of thelyophilisation trial are summarized in table 4.

TABLE 4 API:TRIS Bulking agent Bulk solution Cake Formulation molarratio (g/100 ml) pH density (g/mL) Appearance A 1:2  4% 6.70 1.031 Whitecompact F 1:2  2% 6.47 1.024 Partially collapsed L 1:2.1 1% 6.99 1.021Collapsed N 1:2.1 — 7.08 1.017 Collapsed Placebo — 4% 10.88 — Whitecompact

Formulation A gave a cake with the desired characteristics. Thisformulation was reconstituted with 20 ml and 25 ml of water forinjection to check osmolality, obtaining a value of 0.569 osml/kg forthe formulation reconstituted with 20 ml and a value of 0.444 osml/kg ifreconstituted with 25 ml.

Considering the results so far obtained for each formulation,formulation A (with API:trometamol molar ratio 1:2 and mannitol asbulking agent at 4% in solution) was chosen for development.

Additional trials were performed in order to check if it were possibleto decrease the osmolality value by a slight reduction of the bulkingagent, but the cake appearance was not satisfactory as appearedpartially melted.

An additional trial named formulation C (API/trometamol 1:1, mannitol4%) was performed maintaining the same ratio of API/excipients by aadding the remaining amount of trometamol in the solution ofreconstitution in order to optimize and reduce the length of the freezedrying process.

The process parameters and conditions applied for manufacturingformulation A and formulation C are listed in the “Methods” section.

Formulation A and formulation C were further subjected to a HPLCstability test under stressed conditions at 60° C. after 24 and 72hours; the HPLC method is described in the “Methods” section. Theresults of the HPLC tests, which are summarized in table 5, showed thatformulation A is more stable and presents a lower percentage ofimpurities than formulation C.

TABLE 5 Formulation A Formulation C TEST T0 24 h 72 h T0 24 h 72 h Assay(mg/vial) 495.5 486.8 482.6 504.7 493.3 467.5 Assay % nominal 99.1 97.496.5 100.9 98.7 93.5 Water content % * 2.8 1.6 n.a. 0.6 0.6 n.a.Impurity/ RRT Area % Area % Area % Area % Area % Area % Relatedsubstance *RF *RF *RF *RF *RF *RF Glutamic acid 0.45-0.47 ND ND 0.34 NDND 0.10 IMP 1 1.95-1.93 0.01 0.08 0.26 0.10 0.89 2.31 IMP 2 2.24-2.210.00 0.02 0.08 0.01 0.06 0.17 IMP 5 2.30-2.33 0.02 0.00 0.00 0.02 0.000.00 *Karl Fischer coulometer, model 684 KF (Metrohm) or equivalentLegend: RRT = relative retention time: time of peak elution compared tothe time of eluition of the main peak (carglumic acid). RF = responsefactor (factor applied to correctly quantify the amount of theimpurities) ND = not detected (peak below the limit of detection of theHPLC method)

Based on the results of the development activities performed,formulation A was considered the most appropriate and then selected forfurther development.

As reported above, during the freeze drying cycle optimization,different trometamol:API molar ratios were tried so to speed up theprocess and to optimise the solid state properties as well. Both thecarglumic acid:trometamol 1:1 ratio and the 1:2 one showed goodmanufacturability in terms of process. Despite of this, the furtheraccelerated stability study described above indicates that theformulation with the ratio 1:2 has an improved stability profile ifcompared to the 1:1 molar ratio. Surprisingly, due to the hygroscopiccharacter of carglumic acid, although the water content of the 1:2formulation is higher than the one detected in the 1:1 formulation, thechemical stability is better using the 1:2 ratio. Thus, the increasedtrometamol amount seems to protect the API from degradation triggered bythe free water still remaining after process completion.

Stability Study (Technical Batch Under ICH Conditions):

After completion of the development work, a stability study under ICHconditions was also performed to gather data about the long-term(commercial) stability of the selected formulation A. The resultsobtained up to 12 months with a vial of lyophilized formulation Acontaining 500 mg of carglumic acid stored at 2-8° C. are summarised intable 6.

TABLE 6 Lyophilized Drug Product Results Test T0 1 Month 2 Months 3Months 6 Months 9 Months 12 Months Appearance Freeze- UnchangedUnchanged Unchanged Unchanged Unchanged Unchanged dried, white, compactcake Assay mg/vial 504.7 500.4 504.1 496.6 498.2 510.1 503.1 Assay %100.9 100.1 100.8 99.3 99.6 102.0 100.6 Related Substances % (1)(2)Specified Related Substances Glutamic acid ND ND ND ND ND ND ND RRT0.47IMP 6 RRT ND ND ND ND ND ND ND 1.20 IMP 1 RRT <0.10 <0.10 <0.10 <0.10<0.10 <0.10 <0.10 1.93 (0.011) (0.013) (0.008) (0.005) (0.005) (0.006)(0.007) IMP 2 RRT <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 2.21 (0.003)(0.004) (0.004) (0.002) (0.001) (0.001) (0.001) IMP 5a RRT <0.10 <0.10<0.10 <0.10 <0.10 <0.10 <0.10 2.34 (0.015) (0.015) (0.023) (0.019)(0.019) (0.022) (0.020) IMP 5b RRT ND ND ND ND ND ND ND 2.77 Each OtherND ND ND ND ND ND Individual Related Substance Total Related <0.10 <0.10<0.10 <0.10 <0.10 <0.10 <0.10 Substances Water Content 1.4% 1.5% 1.3%1.4% 1.4% 1.3% 1.5% Reconstituted solution in Sterile Water forInjections Results T0 1 Month 2 Months 3 Months 6 Months 9 Months 12Months Test (3) (3) (3) (4) (4) (4) (4) Reconstitution ≈1 min ≈1 min ≈1min ≈1 min ≈1 min ≈1 min ≈1 min Time Appearance of Clear, UnchangedUnchanged Unchanged Unchanged Unchanged Unchanged Reconstitutedsolution, Solution free from visible particles pH of 6.3 6.3 6.3 6.4 6.36.3 6.3 Reconstituted Solution Note (1): The results <0.10% (LOQ) arereported in brackets only for information. Note (2): The Total RelatedSubstances % is the sum of the reportable (≥0.10%) specified andunspecified impurities. Note (3): The reconstitution volume is 20 mlNote (4): The reconstitution volume is 25 ml

The results obtained up to 12 months with a vial of lyophilizedformulation A containing 500 mg of carglumic acid stored at 25° C./60%RH are summarised in table 7.

TABLE 7 Lyophilized Drug Product Results Test T0 1 Month 2 Months 3Months 6 Months 9 Months 12 Months Appearance Freeze Unchanged UnchangedUnchanged Unchanged Unchanged Unchanged dried, white compact cake Assay504.7 495.8 508.3 498.5 499.1 509.1 503.0 mg/vial Assay % 100.9 99.2101.7 99.7 99.8 101.8 100.6 Related Substances % (1)(2) SpecifiedRelated Substances Glutamic ND ND ND ND ND ND ND acid RRT 0.47 IMP 6 RRTND ND ND ND ND ND ND 1.20 IMP 1 RRT <0.10 <0.10 <0.10 <0.10 <0.10 <0.10<0.10 1.93 (0.011) (0.012) (0.029) (0.017) (0.030) (0.050) (0.055) IMP 2RRT <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 2.21 (0.003) (0.004)(0.011) (0.005) (0.007) (0.011) (0.015) IMP 5a RRT <0.10 <0.10 <0.10<0.10 <0.10 <0.10 <0.10 2.34 (0.015) (0.022) (0.021) (0.021) (0.021)(0.019) (0.021) IMP 5b RRT ND ND ND ND ND ND <0.10 2.77 (0.012) EachOther Indiv. Relat. Substance UNK RRT ND ND ND ND <0.10 <0.10 <0.10 0.63(0.050) (0.046) (0.051) UNK RRT ND ND ND ND <0.10 <0.10 <0.10 0.87(0.044) (0.059) (0.050) UNK RRT ND ND ND ND <0.10 <0.10 <0.10 1.15(0.032) (0.050) (0.082) Total <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10Related Substances Water 1.4% 1.4% 1.3% 1.3% 1.3% 1.4% 1.3% ContentReconstituted solution in Sterile Water for Injections Results 2 Months3 Months 6 Months 6 Months 12 Months Test T0 (3) 1 Month (3) (3) (4) (4)(4) (4) Reconstitution ≈1 min ≈1 min ≈1 min ≈1 min ≈1 min ≈1 min ≈1 minTime Appearance Clear Unchanged Unchanged Unchanged Unchanged UnchangedUnchanged of solution Reconstituted free from Solution visible particlespH Recon. 6.3 6.3 6.3 6.3 6.3 6.3 6.3 Sol. Note (1): The results <0.10%(LOQ) are reported in brackets only for information. Note (2): The TotalRelated Substances % is the sum of the reportable (≥0.10%) specified andunspecified impurities. Note (3): The reconstitution volume is 20 mlNote (4): The reconstitution volume is 25 ml

All the chemical and physical results obtained so far are fully matchingthe specifications required for commercial purposes at both 2-8° C. and25° C. storage conditions.

Methods

Manufacturing Method:

The freeze drying cycle applied for the manufacturing of bothformulation A and formulation C is described below.

Batch size: 2 liters batch

Bulk solution was filled into one tray of 31/39 vials with 20 ml fillingvolume. One tray was filled with mannitol solution to fully load thefreeze-drier and the product lyophilized according to the lyophilizationcycle described in table 8.

TABLE 8 Shelves T Set Time Actual time Lyophilization Cycle Set point (°C.) (hrs:min) (hrs:min) Freezing (shelves cooling) −42° C. 01:00 03:00Freezing (shelves holding) −42° C. 06:00 06:00 Annealing (shelvesheating)  −5° C. 01:20 01:00 Annealing (shelves holding)  −5° C. 04:0004:00 Freezing (shelves cooling) −40° C. 00:35 02:00 Freezing (shelvesholding) −40° C. 02:00 03:00 Primary drying (shelves heating) −10° C.05:00 05:00 Primary drying (shelves holding) −10° C. 48:00  55:00*Secondary drying (shelves heating) +35° C. 03:45 04:00 Secondary drying(shelves holding) +35° C. 10:00 10:00 Secondary drying (shelves heating)+45° C. 00:10 01:00 Secondary drying (shelves holding) +45° C. 10:0010:00 Secondary drying (shelves cooling) +25° C. 00:20 01:00 Secondarydrying (shelves holding) +25° C.  24:00** 15:00 Chamber pressure: 200μbar Stoppering under partial vacuum: 700 mbar — — Total cycle duration121 *= time needed for the product to reach set up T ° C. = −23° C. tostart secondary drying **= time adjustable (min 2 hours) to unload thefreeze drier during working hours

At the end of the cycle, the vials were stopped under partial nitrogen(700 mbar) within the freeze-drier chamber and sealed with flip-offcaps.

HPLC method:

Materials and Reagents

Carglumic acid Reference Standard

Deionized water, Milli Q grade or equivalent

Methanol, HPLC grade

KH₂PO₄, ACS Reagent

H₃PO₄ 85%, ACS Reagent

Equipment

HPLC system Agilent 1100 series or equivalent equipped with UV-VISdetector, cooled auto sampler, degassing system and column oven

Acquisition Data System

HPLC column Develosil 5 μm, RPAQUEOUS-AR C30, 250×4.6 mm or equivalent

Pre-column Gemini C18 or equivalent

Balance accurate to 0.001 mg

High precision laboratory glassware

Chromatographic Conditions

Column temperature: 25° C.

Mobile phase A: KH₂PO₄ 50 mM pH 2.0 per H₃PO₄ 85%

Mobile hase B: CH₃OH

Flow rate: 1.0 ml/min

Injection volume: 50 μl

Autosampler temperature: 5° C.

Detection wavelength: UV at 215 nm

Elution mode: Gradient as reported in table 9

TABLE 9 Mobile Mobile TIME (min) phase A % phase B % 0 100 0 8 100 0 1490 10 28 90 10 30 100 0 40 100 0 Run Time 40 minutes

Under these conditions the retention time (Rt) of carglumic acid isabout 6.6 min. Slight variations of the mobile phase composition and theflow rate may be carried out to provide a suitable elution time and tomeet the requirements of the SST

1. A pharmaceutical formulation comprising carglumic acid or apharmaceutically acceptable salt or derivative thereof and a bufferingagent having a pK_(a) from 5.5 to 9.0 at 25° C.
 2. The pharmaceuticalformulation according to claim 1, wherein the buffering agent istrometamol.
 3. The pharmaceutical formulation according to claim 2,wherein the carglumic acid:trometamol molar ratio is from 1:1 and 1:2.5.4. The pharmaceutical formulation according to claim 1 furthercomprising at least one bulking agent.
 5. The pharmaceutical formulationaccording to claim 4, wherein the bulking agent is mannitol.
 6. Thepharmaceutical formulation according to claim 4, wherein the carglumicacid:bulking agent weight ratio is from 25:32 and 25:50.
 7. Thepharmaceutical formulation according to claim 5 comprising carglumicacid and mannitol, wherein the buffering agent is trometamol, thecarglumic acid:trometamol molar ratio is from 1:1 and 1:2.5, and thecarglumic acid:mannitol weight ratio is from 25:32 and 25:50.
 8. Thepharmaceutical formulation according to claim 1, wherein the formulationis.
 9. The pharmaceutical formulation according to claim 1, wherein theformulation is a water solution.
 10. The pharmaceutical formulationaccording to claim 9, wherein a carglumic acid concentration is higherthan 2% w/v.
 11. AThe pharmaceutical formulation according to claim 1,further comprising one or more physiologically acceptable excipients.12. A method for manufacturing a pharmaceutical formulation according toclaim 8, said method comprising freeze-drying a pharmaceuticalformulation which is a water solution comprising carglumic acid or apharmaceutically acceptable salt or derivative thereof, and a bufferingagent having a pK_(a) from 5.5 to 9.0 at 25° C.
 13. A method fortreating hyperammonaemia, comprising administering the pharmaceuticalformulation according to claim 1 to a patient in need thereof.
 14. Themethod for treating hyperammonaemia according to claim 13, wherein thepharmaceutical formulation comprises from 400 to 600 mg of carglumicacid, and the patient is an adult.
 15. The method for treatinghyperammonaemia according to claim 13, wherein the pharmaceuticalformulation comprises from 25 to 200 mg of carglumic acid, and thepatient belonging belongs to the paediatric population.
 16. The methodfor treating hyperammonaemia according to claim 13, wherein thepharmaceutical formulation is administered parenterally.